Project summary In nature, bacteria often form complex communities that enable them to adapt to their environment and to carry out particular functions. For example, the community of bacteria in the human gut in uences a variety of aspects of health and disease, while some infections are characterized by the formation of lms of living cells (bio lms). These bio lms have been extensively investigated in medical and industrial contexts, but the biophysical rules underlying these living communities have remained unclear. To close this gap, the proposed investigations will develop genetic constructs, bacterial strains, visu- alization tools, and biophysical models in order to set the stage for the rational engineering of complex microbial communities that carry out de ned functions. Speci cally, the investigators propose to (1) develop quantitative experimental tools to optically pattern single-species and multispecies bio lms; and (2) through biophysical modeling and experimentation, investigate the structural development of ecolog- ically interacting consortia from initial seeding. Critically, the proposed investigations will establish and validate a new platform, bio lm lithography, that will enable major advances in the use of optogenetics and synthetic biology for bioengineering, research and therapeutic purposes. This broadly applicable platform will answer two pairs of crucial questions in the proposed inves- tigations. 1a) Can the light-activated expression of bio lm genes yield robust, high-resolution spatial patterning of bio lms on a 2D surface? 1b) Can the platform be used to control multiple genes and populations in parallel by using multichromatic light stimuli? 2a) Can these tools be used to generate, study, and understand stable, spatially patterned multispecies bio lm consortia? 2b) Are experimental measurements of bio lm dynamics supported by quantitative biophysical models? Taken together, the combination of theory and experiment proposed here will set the stage for the plug-and-play design of microbial communities with both complex structure and function. These advances will signi cantly lower access barriers to complex synthetic biology, driving innovation across elds as well as across socioeconomic divisions. When coupled with the future rational design of cellular genomes and structures, our platform for the construction and manipulation of light-patterned living communities has the potential to signi cantly advance medicine and also material sciences.